lable at ScienceDirect

Soil Biology & Biochemistry 81 (2015) 159e167

Contents lists avai

Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Responses of enzymatic activities within soil aggregates to 9-yearnitrogen and water addition in a semi-arid grassland

Ruzhen Wang a, f, Maxim Dorodnikov b, Shan Yang a, c, Yongyong Zhang a, f,Timothy R. Filley d, Ronald F. Turco e, Yuge Zhang c, Zhuwen Xu a, Hui Li a, Yong Jiang a, *

a State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, Chinab Soil Science of Temperate Ecosystems, Büsgen-Institute, Georg August University of G€ottingen, Büsgenweg 2, 37077 G€ottingen, Germanyc Key Laboratory of Regional Environment and Eco-remediation, College of Environment, Shenyang University, Shenyang 110044, Chinad Department of Earth, Atmospheric, and Planetary Sciences, Purdue University, West Lafayette, IN 47907, USAe Department of Agronomy, Purdue University, West Lafayette, IN 47907, USAf University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

Article history:Received 18 July 2014Received in revised form29 October 2014Accepted 18 November 2014Available online 29 November 2014

Keywords:Global changeNitrogen depositionPrecipitation regimesExtracellular enzymesMicrobial nitrogen limitationTemperate grassland

* Corresponding author. Tel.: þ86 24 83970902; faE-mail address: jiangyong@iae.ac.cn (Y. Jiang).

http://dx.doi.org/10.1016/j.soilbio.2014.11.0150038-0717/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Soil microorganisms secrete enzymes used to metabolize carbon (C), nitrogen (N), and phosphorus (P)from the organic materials typically found in soil. Because of the connection with the active microbialbiomass, soil enzyme activities can be used to investigate microbial nutrient cycling including the mi-crobial response to environmental changes, transformation rates and to address the location of the mostactive biomass. In a 9-year field study on global change scenarios related to increasing N inputs (ambientto 15 g N m�2 yr�1) and precipitation (ambient to 180 mm yr�1), we tested the activities of soil b-glucosidase (BG), N-acetyl-glucosaminidase (NAG) and acid phosphomonoesterase (PME) for three soilaggregate classes: large macroaggregates (>2000 mm), small macroaggregates (250e2000 mm) andmicroaggregates (<250 mm). Results showed higher BG and PME activities in micro-vs. small macroag-gregates whereas the highest NAG activity was found in the large macroaggregates. This distribution ofenzyme activity suggests a higher contribution of fast-growing microorganisms in the micro-comparedwith the macroaggregates size fractions. The responses of BG and PME were different from NAG activityunder N addition, as BG and PME decreased as much as 47.1% and 36.3%, respectively, while the NAGincreased by as much as 80.8%, which could imply better adaption of fungi than bacteria to lower soil pHconditions developed under increased N. Significant increases in BG and PME activities by as much as103.4 and 75.4%, respectively, were found under water addition. Lower ratio of BG:NAG and higherNAG:PME underlined enhanced microbial N limitation relative to both C and P, suggesting the repressionof microbial activity and the accompanied decline in their ability to compete for N with plants and/or theaccelerated proliferation of soil fungi under elevated N inputs. We conclude that changes in microbialactivities under increased N input and greater water availability in arid- and semi-arid grassland eco-systems where NPP is co-limited by N and water may result in substantial redistribution of microbialactivity in different-sized soil particles. This shift will influence the stability of SOM in the soil aggregatesand the nutrient limitation of soil biota.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Extracellular enzymes are secreted by soil microorganisms tomineralize organic carbon (C), nitrogen (N), and phosphorus (P)from soil organic matter (Waring et al., 2014). Extracellular activ-ities are distinct from intracellular ones as they can be stabilized by

x: þ86 24 83970300.

abiotic soil components (Dilly and Nannipieri, 1998). Measuredenzyme activities represent the apparent catalytic history of a soilas continuously modified by soil microorganism in response toenvironmental changes (Dilly and Nannipieri, 2001). As a result,enzyme activities can be used to assess microbial nutrient demands(Schimel and Weintraub, 2003; Moorhead and Sinsabaugh, 2006)and used to formulate an ecosystem response index that reflectenvironmental changes (Ajwa et al., 1999; Sinsabaugh et al., 2008).For example, b-glucosidase (BG) has been used to assess the mi-crobial response to long-term N amendments in a tall-grass prairie

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167160

soil (Ajwa et al., 1999) and N-acetyl-glucosaminidase (NAG) hasbeen utilized to quantify N-limitation effecting woody plantencroachment into grasslands (Creamer et al., 2013). Enzymaticprocess stoichiometry is suggested as a means to understand the Cand N limitations in soil processes as it is demonstrated that soilenzymatic activities and stoichiometry are related to substrateavailability, soil pH, and climatic factors (e.g., precipitation andtemperature) (Sinsabaugh et al., 2008, 2009; Waring et al., 2013).However, the effects of environmental factors, such as elevated Ninputs and precipitation on enzymatic stoichiometry are unclear.Various global change scenarios have suggested that increased in-puts of reactive N from fertilization and fossil fuel combustion andaltered precipitation regimes will become common (Knapp et al.,2002; Liu et al., 2009, 2013). As soil enzyme activities are sensi-tive to ecosystem fluctuations, they can serve as indicators ofvarious responses of the plant-soil system to changes in N depo-sition (Sinsabaugh et al., 2005), elevated atmospheric CO2(Dorodnikov et al., 2009a,b), and precipitation intensity (Henryet al., 2005; Bardgett et al., 2008; Pendall et al., 2008).

Clearly, nitrogen and water availability are two driving factorsaffecting grassland net primary productivity (NPP) (Xu et al., 2012),especially in semi-arid grasslands where evaporation greatly ex-ceeds annual precipitation inputs (Heisler-White et al., 2008).Recent grassland-related field studies in Inner Mongolia suggesteda plant NPP of about 1.5 tons ha�1 is both N- and water-limited,because N addition above 5.25e17.5 g N m�2 yr�1 of backgroundincreased NPP by 13%e62 % (Bai et al., 2010), while water additionincreased above- and belowground NPP by 32.9% and 38.3%,respectively (Xu et al., 2010). However, soil microorganisms are notlimited by the same factors that limit plant systems (Hobbie et al.,2005; Wei et al., 2013). For example, Wei et al. (2013) reporteddifferent N saturation levels (threshold levels for N demand) forplants and soil microorganism highlighting that microbes can belimited by C or P while plants are N limited (Treseder, 2008).Additionally, reduction in both the size and activity of soil microbialbiomass were shown under higher N availability in temperategrasslands (Gutknecht et al., 2012;Wei et al., 2013), which indicatesmicroorganisms are not always limited by N. On the other hand,positive effects of added N were also observed (Zeglin et al., 2007;Keeler et al., 2009). Keeler et al. (2009) found N addition to increasethe activity of phosphatase and cellobiohydrolase by 13% and 17%,respectively. Similar findings were also reported by Zeglin et al.(2007) where N increased both cellulolytic activities (BG and cel-lobiohydrolase) and phosphatase activity. Other studies showedthat added water in grassland ecosystems stimulated (Zhou et al.,2013) or suppressed microbial activity (Henry et al., 2005)depending on the study site. For instance, increased water avail-ability resulted in increases of NAG, leucine aminopeptidase, andalkaline phosphomonoesterase (PME) in an Inner Mongolia grass-land (Zhou et al., 2013), while water addition resulted in decreasesof BG, NAG, and PME in a California grassland soil (Henry et al.,2005).

Aggregate structure can affect microbial activities as fluxes ofwater and oxygen (Six et al., 2004) and accessibility of SOM willdiffer between aggregate-size classes (Jastrow et al., 2007). Jastrowet al. (2007) report most labile SOM is concentrated in macroag-gregates and more recalcitrant, or less accessible SOM is resident inmicroaggregates resulting in overall higher enzyme activities inmacro-vs. microaggregates (Dorodnikov et al., 2009b). The study ofsoil microbial enzyme activities on an aggregate level could provideinsight into soil C and N cycling in response to increased N inputand precipitation.

The objectives of this study were to examine the effects ofelevated N inputs and precipitation intensity on the distributionand activity of C-, N-, and P-acquiring enzymes by evaluating

aggregate size fractions for soils collected from the semi-aridgrasslands of Inner Mongolia, China. A prior field manipulationexperiments, had demonstrated significant increase of NPP inresponse to four 4-year N and water addition (Xu et al., 2010) andSOC in response to 7-year water addition (Wang et al., 2014). Wehypothesized that (i) microbial biomass and enzyme activitieswould increase in macroaggregates because of presumably higheramount of labile SOM; (ii) N- acquiring enzymes would respond toN additions in a way that is different from C- and P-acquiring en-zymes because N addition would decrease the substrate C:N ratioand increase the N:P ratio; and (iii) increasing moisture inputs forecosystems under water limitation should stimulate microbial ac-tivity resulting in higher overall enzyme production for C-, N-, andP-acquisition. As N amendment may potentially cause C and Plimitation, we predict that the ratio of b-glucosidase to N-acetyl-glucosaminidase will increase while N-acetyl-glucosaminidase tophosphomonoesterase ratio will decrease under higher Navailability.

2. Materials and methods

2.1. Field site and experimental design

The study site is located in Duolun County, Inner Mongolia innorthern China (116�170 E and 42�020 N, elevation 1324m a.s.l.). Themean annual temperature is 2.1 �C with mean monthly tempera-ture ranging from �17.5 �C in January to 18.9 �C in July, and themean annual precipitation is 379.4 mm with approximately 86%occurring fromMay to September. The plant community at the siteis a typical temperate grassland dominated by prairie sagewort(Artemisia frigidaWilld.), wheatgrass (Agropyron cristatum Gaertn.),and needlegrass (Stipa krylovii Roshev.). The soil type is classified asHaplic Calcisols according to the FAO classification with 63% sand,20% silt, and 17% clay, respectively (Liu et al., 2009).

In April 2005, a split-plot experiment design was applied to thesite. Twelve 8 m � 8 m plots were established in each treatmentblock (107 m � 8 m) with 1 m buffer zone between any twoadjacent plots; each block was replicated seven times. The blockswere divided into two main plots based on water treatment(ambient precipitation and 180 mm of water addition) and theneach main plot was divided into six subplots. The 180 mm of wateraddition is an approximately 50% increase above mean annualprecipitation based on meteorological record for the site (Xu et al.,2012). This experiment is part of an on-going project designed toinvestigate the effect of increased N and water on ecosystem re-sponses in the Inner Mongolia grassland (Xu et al., 2010, 2012). TheN addition plots were randomly assigned to subplots within eachmain plot. The water addition plots received 15mm of precipitationweekly by sprinkling irrigation during the growing season (12consecutive weeks from June to August). Nitrogen (in the form ofurea) was applied at four levels: 0 (CK), 5 g N m�2 yr�1 (N5),10 g N m�2 yr�1 (N10), and 15 g N m�2 yr�1 (N15) in four of sixsubplots, half of which was applied in early May and the other halfin late June from 2005 to 2013. Background N inputs (atmosphericdeposition plus fertilizer application) in this area are about5 g m�2 yr�1 so this manipulation represents a 5e10 g m�2 yr�1

increase in N addition.

2.2. Soil aggregate-size fractionation and other soilphysicochemical properties

In September 2013, a composite soil sample from the top0e10 cm soil layer was taken from five randomly selected locationsat each plot from four out of seven blocks in both the N and watertreatment main plots using a 5-cm diameter corer. Fresh soil

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167 161

samples were placed in hard plastic containers to maintain theirprimary structures during transportation to the laboratory. Tominimize the disruption in microbial communities and activities,soil aggregates were isolated by modified dry-sieving method ac-cording to Dorodnikov et al. (2009a,b). The field moisture of thesesoil samples was optimal (10e15%) for dry-sieving which allowedlimited mechanical stress to induce maximum brittle failure alongthe natural planes of weakness. The recovered soil was gentlysieved through a 5 mm screen and visible plant residues and stoneswere removed. The recovered soil samples (500 g) were transferredto a nest of sieves (2000 and 250 mm) on a Retsch AS200 Control(Retsch Technology, Düsseldorf, Germany). The sieves were me-chanically shaken (amplitude 1.5 mm) for 2 min to separate theaggregates >2000 mm (large macroaggregates class), 250e2000 mm(small macroaggregates), and <250 mm (microaggregates class).Samples were used immediately for inorganic nitrogen and dis-solved organic carbon (DOC) analyses and frozen at �20 �C for useon enzyme assays.

Soil moisture of the separated aggregates was determined asmass loss after drying the isolates at 105 �C for 24 h. Nitrate andammonium were determined colorimetrically from 1 M KCl soilextracts from the fresh soil aggregate samples using an Auto-AnalyserⅢ continuous Flow Analyzer (Bran& Luebbe, Norderstedt,Germany). Additionally, soil pH was determined in a 1:5 (w/v) soil-to-water extract of soil aggregate samples from treatments andcontrols with a PHS-3G digital pH meter (Precision and ScientificCorp., Shanghai, China). Soil DOC was extracted by adding 50 ml of0.5 M potassium sulfate (K2SO4) to subsamples of 25 g homoge-nized soil fractions, and agitated on an orbital shaker at 120 rpm for1 h (Wei et al., 2013). After filtering through 0.45 um cellulose ac-etate filter paper, the filtrate was analyzed by a TOC analyzer (HighTOC, Elementar).

2.3. Microbial biomass C analysis and enzyme assays

Microbial biomass C (MBC) was determined using thefumigation-extraction method (Vance et al., 1987). Briefly, 10 g (dryweight equivalent) of each soil aggregate fraction was fumigatedwith ethanol-free chloroform (CHCl3) for 24 h at 25 �C. Simulta-neously, another subsample was kept at the same conditionswithout fumigation. After complete removal of CHCl3, organic Cfrom fumigated and unfumigated soil samples were extracted with0.5 M K2SO4 with a soil:extractant ratio of 1:4 (w/v) and shaken at150 rpm for 1 h. After filtration with Whatman no. 2v filter paper,extractable organic C in soil extracts was analyzed by a TOCanalyzer (High TOC, Elementar). Microbial biomass C was calcu-lated as the difference between fumigated and non-fumigatedsamples and normalized to the weight of the soil fraction. To cor-rect for incomplete extraction, we used efficiency factor of 0.38(Vance et al., 1987) to calculate the actual MBC concentration asdescribed by Zhang et al. (2013a) for the soils in this area.

Frozen (at �20 �C) field moist soil aggregate samples werethawed to 4 �C one week prior to the start of any enzyme assay. Themeasurement of activities of b-glucosidase (BG), N-acetyl-b-D-glu-cosaminidase (NAG) and acidic phosphomonoesterase (PME) wasperformed on the basis of p-nitrophenol (PNP) released aftercleavage of enzyme-specific synthetic substrates according tomethod of Tabatabai (1994). The specific substrates (Sigma, St.Louis, USA) were p-nitrophenyl-b-D-glucopyranoside for BG, p-nitrophenyl-N-acetyl-b-D-glucosaminide for NAG and p-nitro-phenyl-phosphate for acid phosphomonoesterase (PME). For BGactivity, 1.0 g of soil aggregates was weighed into Erlenmeyer flaskto mix with a pH 6.0 modified universal buffer consisting of 0.1 Mtrihydroxymethyl aminomethane, 0.067 M citric acid monohydratecompound, and 0.1 M boric acid. The indicator substrate, 0.05 M p-

nitrophenyl-b-D-glucopyranoside was added to the reaction systemfollowed by 1 h incubation. Reactions were stopped by adding0.5 M CaCl2 and 0.1 M trihydroxymethyl aminomethane which wasbuffered to pH 12. Controls were performed with the substratebeing added after the reactions were stopped. The products werefiltered through Whatman no. 2v filter paper and measuredcolorimetrically at 410 nm (Tabatabai, 1994) with a UV-VIS spec-trophotometer (UV-1700, Shimadzu). The procedures for the assaysof NAG and PME activities were the same as for BG except using p-nitrophenyl-N-acetyl-b-D-glucosaminidine and p-nitrophenyl-phosphate as the substrate and buffering pH of reaction systems to5.5 (Parham and Deng, 2000) and 6.5 (Tabatabai, 1994), respec-tively. The activities of BG, NAG and PMEwere expressed inmg PNPreleased per kg dry soil fraction per hour.

2.4. Statistical analysis

ANOVAswith a split-plot designwere executed to determine theeffect of water (between-subject), N addition and soil aggregatesizes (within subject) and their interactions on the pH values, theconcentrations of MBC and DOC, the activities of BG, NAG and PMEand the ratios of BG:NAG, BG:PME and NAG:PME. The effects of Naddition rates on the activities of BG, NAG and PME and the ratio ofBG:NAG, BG:PME and NAG:PME were determined by one-wayANOVA and run separately for ambient precipitation and wateraddition. Pearson correlation analysis was used to examine therelationship among soil parameters. All statistical analyses wereperformed in SPSS 16.0 (SPSS, Inc., Chicago, IL, U.S.A) and statisticalsignificance was accepted at P < 0.05.

3. Results

3.1. Soil pH, DOC and MBC values among soil aggregates

Soil pH decreased significantly (P < 0.05) with increasing Ninput under either normal or elevated water (Fig. 1a). Water addi-tions (W) significantly increased soil pH in N5, N10 and N15 treat-ments of large-macroaggregates and microaggregates, and in N5treatment of small macroaggregates (Fig. 1a). Neither aggregatesize nor interactive N,Wand aggregate size (A) effects was detectedin changes of soil pH (Table S1). The DOC concentration was notaffected by water addition or soil aggregate sizes but significantlyincreased by the highest N (N15) for both large- and small-macroaggregates and by N additions at N5 and N15 for micro-aggregates under high level of water conditions (Table S1, Fig. 1b).

Under normal level of precipitation, N addition significantlydecreased MBC in small macroaggregates for the N5 and N15treatments and in the microaggregates for N15 treatments. Incontrast to normal level of precipitation, under high levels of pre-cipitation, MBC was reduced in the N15 treatment for large mac-roaggregates (Fig. 1c). Water addition significantly increased MBCin N5 of small macroaggregates (Fig. 1c). The concentration of MBCin aggregates increased as follows: small macroaggregates < largemacroaggregates < microaggregates.

3.2. Response of enzyme activities in different soil aggregate classesto N and water addition

In general, soil b-glucosidase activity was affected by water andN treatments, and was different across the aggregate fractions;there was a significant W � N and W � A interaction (Table S1).Across the three aggregate fractions, the activity of BG decreasedsignificantly (P < 0.05) at the highest N addition rate (N15) by31.5e47.1 % as compared to respective control plots under normal-level water conditions (Fig. 2a). Under elevatedwater inputs, the BG

Fig. 1. Soil pH values (a) and concentrations of soil dissolved organic carbon (DOC) (b) and microbial biomass carbon (MBC) (c) in three soil aggregate-size classes of the control (CK)and three N addition treatments (g m�2 yr�1) under ambient and elevated water levels. Values are means of four replicates (±SE). Letters indicate significant differences amongmeans for the N addition without or with water addition separately (lowercase letters) and differences among soil aggregate-size classes (capital letters). Asterisks indicate sig-nificant difference between water treatments for each N and CK treatment.

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167162

activity was significantly higher within each N treatment and soilfraction as compared to normal level of water conditions by45.5e103.4 %, however the decrease of BG activity with increasingN supply was much more pronounced (Fig. 2a). The BG activity wasdistributed differently through aggregate-size classes: undernormal level of water regime the lowest activity was measured insmall macroaggregates followed by comparable activities in largemacro- and microaggregates; under higher level of water treat-ment, however, BG activity increased in the order large macro-> micro- > small macroaggregates with the highest being CK(without N additions) (Fig. 2a).

Our results showed that there were significant N, aggregate size,W � N, and N � A effects on N-acetyl-b-D-glucosaminidase activity,whereas no significant water, W � A, and W � N � A effects werefound (Table S1). N addition significantly increased the NAG activityin the three soil aggregates by as much as 80.8%, except for smallmacroaggregate and microaggregates under normal-level waterconditions (Fig. 2b). The NAG activity was significantly higher inwater addition plots of N5 in large macroaggregates and of N15 inmicroaggregates while it was lower for N15 in large macroaggre-gates as compared to normal-level water conditions (Fig. 2b).

Across three soil fractions, the NAG activity in large macroaggre-gates was the highest under N treatments of N10 and N15 for bothwater treatments.

While water treatment substantially stimulated phospho-monoesterase activity, N addition significantly decreased PME ac-tivity under both water regimes throughout three soil aggregateclasses by 0.8e36.3 % (Fig. 2c). Among three aggregate-size classes,the PME activity was the highest in microaggregates followed bylarge macro- and small macroaggregates. This trend was especiallypronounced under high-level water treatment (Fig. 2c).

3.3. Response of enzymatic ratios to N and water addition amongsoil aggregate classes

The ratio of b-glucosidase: N-acetyl-b-D-glucosaminidase wassignificantly decreased by N addition under both normal and highlevel of water treatments (Table S1 vs. Fig. 3a). Water additionincreased (P < 0.05) the ratio of BG:NAG across the three soilaggregate fractions (Fig. 3a). Aggregate sizes significantly influ-enced the distribution of BG:NAG ratio (Table S1). This ratio was thehighest in microaggregates under normal-level water treatment

Fig. 2. Soil enzyme activities of b-glucosidase (BG) (a), N-acetyl-b-D-glucosaminidase (NAG) (b) and acid phosphomonoesterase (PME) (c) in three soil aggregate-size classes of thecontrol (CK) and three N addition treatments (g m�2 yr�1) under ambient and elevated water levels. Values are means of four replicates (±SE). Letters indicate significant differencesamong means for the N addition without or with water addition separately (lowercase letters) and differences among soil aggregate-size classes (capital letters). Asterisks indicatesignificant difference between water treatments for each N and CK treatment.

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167 163

and with increased N additions. However, under N control treat-ment and high level of precipitation, the highest BG:NAG ratio wasdetected in large macroaggregates (Fig. 3a). Significant W � N, andN � A effects were detected on the ratio of BG:NAG (Table S1). Theratio of BG:NAG ranged from 4.6 to 14.2 under normal level ofprecipitation conditions and from 8.7 to 26.9 under water additiontreatments throughout three aggregate classes (Fig. 3a).

The highest value of b-glucosidase: phosphomonoesterase ratiowas observed under high level of water supply of CK treatment inlarge macroaggregates (Fig. 3b). Addition of water significantlyincreased BG:PME ratio in CK of small macroaggregates, and N5 andN15 of microaggregates, whereas other differences were not sig-nificant (Table S1, Fig. 3b). Elevated N significantly increased theNAG:PME ratio in three soil aggregate-size classes under bothambient precipitation and water addition treatments (Fig. 3c).Water addition significantly decreased the ratio of NAG:PME in alltreatments of large macroaggregates, in CK and N10 of small mac-roaggregates, and in CK, N5, and N10 of microaggregates (Fig. 3c).Significant interaction of N with water or aggregate size wasdetected on NAG:PME ratio (Table S1).

3.4. Correlations between soil chemical and biologicalcharacteristics

Soil pH had significant positive correlations with MBC, BG ac-tivity, and PME activity (Table S2) while it had significant negativecorrelations with NAG activity (Table S2). MBC positively correlatedwith BG and PME activities but negatively correlated with NAGactivity (Table S2). No significant correlation between DOC andMBC as well as enzyme activities was found (Table S2).

4. Discussion

4.1. Distribution of microbial biomass and enzyme activities amongsoil aggregates

Given that fresher plant material, presumably enriched in labileOM should accumulate in large-size fractions (Jastrow et al., 2007),we expected to observe more accumulation of MBC in the largerfraction. However, we found that MBC was higher in the micro-aggregates than in themacroaggregates (Fig. 1c), which is similar to

Fig. 3. The ratios of BG:NAG (a), BG:PME (b), and NAG:PME (c) in three soil aggregate-size classes of the control (CK) and three N addition treatments (g m�2 yr�1) under ambientand elevated water levels. Values are means of four replicates (±SE). Letters indicate significant differences among means for the N addition without or with water additionseparately (lowercase letters) and differences among soil aggregate-size classes (capital letters). Asterisks indicate significant difference between water treatments for each N andCK treatment.

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167164

the finding reported by Dorodnikov et al. (2009a) and Zhang et al.(2013b). This could be due to the fact that smaller aggregate sizeshave higher specific surface because of a larger portion of clay andsilt to which microbial cells are attached (Amato and Ladd, 1992;Van Gestel et al., 1996). Additionally, smaller pore sizes in micro-aggregates (Jastrow et al., 2007) protect microorganisms frompredation by protozoa or from desiccation (Zhang et al., 2013b) andallow for accumulation in particles with longer mean residencetime.

The distribution of MBC did not coincide with activities of BG,NAG, and PME among the aggregate fractions (Fig. 2). Thus, therelatively higher BG and PME activities in microaggregates versussmall macroaggregates could result from a higher contribution offast-growing microorganisms in microaggregates (Dorodnikovet al., 2009a). Indeed, previous investigations found that BG activ-ity correlated well with fast-growing Gram-negative bacteria and

PME with the whole community (Waldrop et al., 2000). In contrast,Dilly et al. (2001) found microbial mass and BG were representingbetter for integral microbiological characteristics whichmay be dueto the discrepancies between media cultivation and field studies.However, in contrast to BG and PME, and in line with our initialhypothesis, NAG activity was the highest in large macroaggregateswhich is abundant in fresher particulate organic carbon (Fig. 3b). AsNAG activity is thought to be mainly driven by the activity of thefungal community (Miller et al., 1998; Chung et al., 2007), weinterpret the increasing trend of NAG with aggregate size as apreference of habitat by fungi in this system rather than a responseto N availability (Dorodnikov et al., 2009a,b).

In this system, we surmised that a high microbial demand for Prelative to both C and N, and high demand for C relative to N wouldpersist in microaggregates, with higher mineral content, regardlessof N or water treatment. We found this to be the case, evidenced by

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167 165

both BG:PME and NAG:PME ratios in microaggregates being lowerthan in macroaggregates, and the BG:NAG ratio showing anopposite response (Fig. 3). Phosphorus-deficiency of microorgan-isms might derive from P sequestration through mineral in-teractions of clays (Waring et al., 2014), and iron- and aluminum-oxide coatings (Khare et al., 2005). Lower C availability relative toN, as evidenced by lower proportions of extractable DOC to dis-solved inorganic nitrogen (DIN) in microaggregates (Fig. S1), couldexplain the elevated BG to NAG ratio in our study.

4.2. N and water addition induces divergent responses of activitiesof C-,N- and P-acquisition

Consistent with our hypothesis, N addition altered the soil mi-crobial community such that the soil recorded a divergent responseof N- from C- and P- acquisition enzymes (Fig. 2). The activities ofBG and PME, as well as MBC content, were generally repressed byincreased N supply (Figs. 1 and 2). A reduction in overall soil mi-crobial activity under N addition has been documented in both fieldand lab-based studies (Treseder, 2008) although variable responseshave been documented including no net impact (Thomas et al.,2012). When respiration exhibits a suppressed response tochronic N addition, microbial biomass was found to be stronglyrelated to the duration and amount of N added (Treseder, 2008), butnot necessarily dependent on the form of N added (Ramirez et al.,2010). An overall decrease in microbial activity with N was sug-gested to be due to the increase of copiotrophic microorganismsthat rely on more labile C sources, thus relying less on the need forextracellular enzyme secretion (Ramirez et al., 2012). Indeed, in thepresent study we detected positive N effects on dissolved organic Cpool (Fig. 1b), which might lead to an increase in copiotrophicgroups (Fierer et al., 2007). The lack of correlation between DOCand MBC as well as enzyme activities (Table S2) indicated that,overall, the repression of microbial activities was not caused byDOC limitation. Consistent with this conclusion is the increase ofthe NPP/SOC ratio (Fig. S2) under N addition.

Significant positive correlations between soil pH and MBC, BGand PMEwere observed in our study (Table S2). Soil pH is suggestedto be a primary control on microbial activity, enzyme kinetics(Rousk et al., 2009; Wang et al., 2014), and microbial diversity(Fierer and Jackson, 2006). Because soil pH strongly influences thedenaturation of enzyme active center as well as enzyme folding(Frankenberger and Johanson, 1982; Wang et al., 2006), C andnutrient availabilities (Andersson et al., 2000; Kemmitt et al., 2006;Aciego Pietri and Brookes, 2008), and the concentration of DOC(Wei et al., 2013). The negative correlation between NAG and soilpH (Table S2) was consistent with the study of Sinsabaugh et al.(2008) who analyzed global enzymatic database from 40 ecosys-tems. Additionally, the opposite responses of BG and NAG to Naddition, as well as the associated lowering of soil pH, might sug-gest that soil acidification favors fungi over bacteria consistent withpH-fungi relationships (e.g., Miller et al., 1998; Rousk et al., 2009).

The NAG activity may also be induced under a variety of sce-narios that result in soil microbial N limitation, including chemicalsequestration of N through humification in SOM (Creamer et al.,2013) and increases in NPP which transfer N from the soil toplant biomass. This latter case could explain NPP of plants isfrequently limited by N (LeBauer and Treseder, 2008) and as Naddition increased NPP (Xu et al., 2012), and N concentration ofplant biomass at our field site (unpublished data). The activity ofNAG may be induced under a NPP driven microbial N limitation.

In line with our initial hypothesis, water addition significantlyincreased (P < 0.05) MBC and the activities of BG and PME in allthree soil aggregate fractions (Table S1, Fig. 2a and c). Our resultsare consistent with previous surveys in this semi-arid area that

showed positivewater effects onMBC and enzyme activities in bulksoils (Zhou et al., 2013; Zhang et al., 2013a). Other studies havedemonstrated that under improved water conditions, soil nutri-tional compounds were brought into soil solution activating mi-croorganisms that up-regulated enzymatic production (Edwardset al., 2007; Geisseler et al., 2011) which is a likely mechanism toexplain our findings. With the documented increase in plant den-sity and NPP with water addition at this study site (Xu et al., 2010,2012), changes in plant quantity and quality could be indirectdrivers of microbial activity enhancement (Zak et al., 2003; Milcuet al., 2010; Zhang et al., 2013a).

4.3. Responses of enzymatic ratios to elevated N and wateravailability

In contrast to our hypothesis, the ratio of BG:NAG decreasedunder elevated N availability across aggregate classes whileNAG:PME increased (Fig. 3a and c) suggesting N addition elevatedmicrobial demand for N relative to C and P. Higher microbial Ndemand relative to C could result from plants outcompeting mi-crobes for Nwhile allocating N-poor carbon compounds back to therhizosphere (especially mycorrhizal fungi) (Allen, 1991; Tresederand Allen, 2002). Microbial N limitation could also be induced bydeficiency of accessible organic N as heterotrophic microorganismsprefer simple organic N monomers to inorganic N sources(N€asholm et al., 1998; Schimel and Bennett, 2004; Dunn et al.,2006; Creamer et al., 2013). Previous studies suggested that Naddition could induce microbial C limitation through a lower allo-cation by plants to fine root production resulting in less C to the soil(Treseder, 2008). Our conclusion of higher microbial N demandrelative to C, however, was in contrast to the proposed microbial C-limitation cases under N amendment.

Nitrogen addition increased microbial P limitation relative to C(suggested by BG:PME, Fig. 3b). Since new P is derived primarilyfrom rock weathering (Walker and Syers, 1976), it may not keeppace with the supply of C under higher N and water availability(Vitousek et al., 2010). Similar to N limitation, microbial P limitationmay result from increased plant P uptake as revealed in the presentsite by a higher leaf P concentration (unpublished data). In fact, weobserved a decrease in soil aggregate P under N addition (unpub-lished data). Our results suggest that enhanced N inputs haveaccelerated microbial P limitation and could ultimately place apotential constraint on ecosystem productivity at this site.

Significantly higher BG:NAG and BG:PME ratios under higherwater availability suggest a relatively higher microbial C limitationrelative to N and P as compared to normal level of water plots(Waring et al., 2014).With unchanged DOC concentration, however,the increase in microbial growth, discerned from overall positivewater effects on MBC (Table S1), would cause C limitation underincrease water availability. Also, enhanced microbial respirationunder water addition (Niu et al., 2009) might be responsible formicrobial C limitation.

5. Conclusions

After 9-year of N and water field amendment in a semi-aridgrassland, we observed significant changes in soil enzyme activ-ities, physicochemical properties like pH, and MBC among specificsoil size fractions. Overall, microaggregates retained higher BG andPME activities than small macroaggregates suggesting greaterlevels of fast-growing microbes therein regardless of treatment.With N addition, however, BG activity decreased and NAG activityincreased, which was concomitant with a decrease in soil pH; aresponse consistent with a selective proliferation of soil fungi overbacteria. As expected, water addition increased activities of BG and

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167166

PME in all soil fraction sizes, consistent with an up-regulating effectof activated microorganisms under improved water and nutrientconditions that also sparked an increase in NPP. Enzymatic ratiosindicated, however, higher microbial P limitation in micro-aggregates, while higher N limitation under N addition and higherC limitation under water addition over all soil fractions. Underprojected global change scenarios for this region of China, we canexpect changes of microbial activity and chemistry at the multiplespatial scales of the soil continuum that is linked to concomitantchanges in ecosystem NPP. These changes could have importantimpacts related to the localization of microbial community functionin aggregate-size fractions and a change in the nutrient cyclingcapacity of these soils. Overall, identifying how microbes respond(i.e. biomass and functional activity) among soil particles that areresponsible for stabilization of different pools of soil C in systemsunder coupled water-N changes will enhance our capability topredict ecosystem resilience to future global change.

Acknowledgments

We would like to thank Professor Lijun Chen for providingprotocols of enzyme assays and laboratory facilities. We alsoacknowledge support provided by the China-U.S. EcoPartnershipfor Environmental Sustainability. This work was supported by theNational Natural Science Foundation of China (41371251), the Na-tional Key Basic Research Program of China (2011CB403204), andthe Strategic Priority Research Program of the Chinese Academy ofSciences (XDB15010100).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.soilbio.2014.11.015.

References

Aciego Pietri, J.C., Brookes, P.C., 2008. Relationships between soil pH and microbialproperties in a UK arable soil. Soil Biology & Biochemistry 40, 1856e1861.

Ajwa, H.A., Dell, C.J., Rice, C.W., 1999. Changes in enzyme activities and microbialbiomass of tallgrass prairie soil as related to burning and nitrogen fertilization.Soil Biology & Biochemistry 31, 769e777.

Allen, M.F., 1991. The Ecology of Mycorrhizae. Cambridge University Press.Amato, M., Ladd, J.N., 1992. Decomposition of 14C-labelled glucose and legume

material in soils: properties influencing the accumulation of organic residue Cand microbial biomass C. Soil Biology & Biochemistry 24, 455e464.

Andersson, S., Nilsson, S.I., Saetre, P., 2000. Leaching of dissolved organic carbon(DOC) and dissolved organic nitrogen (DON) in mor humus as affected bytemperature and pH. Soil Biology & Biochemistry 32, 1e10.

Bai, Y., Wu, J., Clark, C.M., Naeem, S., Pan, Q., Huang, J., Zhang, L., Han, X., 2010.Tradeoffs and thresholds in the effects of nitrogen addition on biodiversity andecosystem functioning: evidence from inner Mongolia grasslands. GlobalChange Biology 16, 358e372.

Bardgett, R.D., Freeman, C., Ostle, N.J., 2008. Microbial contributions to climatechange through carbon cycle feedbacks. The ISME Journal 2, 805e814.

Chung, H., Zak, D.R., Reich, P.B., Ellsworth, D.S., 2007. Plant species richness,elevated CO2, and atmospheric nitrogen deposition alter soil microbial com-munity composition and function. Global Change Biology 13, 980e989.

Creamer, C.A., Filley, T.R., Olk, D.C., Stott, D.E., Dooling, V., Boutton, T.W., 2013.Changes to soil organic N dynamics with leguminous woody plant encroach-ment into grasslands. Biogeochemistry 113, 307e321.

Dilly, O., Nannipieri, P., 1998. Intracellular and extracellular enzyme activity in soilwith reference to elemental cycling. Journal of Plant Nutrient and Soil Science161, 243e248.

Dilly, O., Nannipieri, P., 2001. Response of ATP content, respiration rate and enzymeactivities in an arable and a forest soil to nutrient additions. Biology andFertility of Soils 1, 64e72.

Dilly, O., Bartsch, S., Rosenbrock, P., Buscot, F., Munch, J.C., 2001. Shifts in physio-logical capabilities of the microbiota during the decomposition of leaf litter in ablack alder (Alnus glutinosa (Gaertn.) L.) forest. Soil Biology & Biochemistry 33,921e930.

Dorodnikov, M., Blagodatskaya, E., Blagodatsky, S., Fangmeier, A., Kuzyakov, Y.,2009a. Stimulation of r-vs. K-selected microorganisms by elevated atmosphericCO2 depends on soil aggregate size. FEMS Microbiology Ecology 69, 43e52.

Dorodnikov, M., Blagodatskaya, E., Blagodatsky, S., Marhan, S., Fangmeier, A.,Kuzyakov, Y., 2009b. Stimulation of microbial extracellular enzyme activities byelevated CO2 depends on soil aggregate size. Global Change Biology 15,1603e1614.

Dunn, R.M., Mikola, J., Bol, R., Bardgett, R.D., 2006. Influence of microbial activity onplant-microbial competition for organic and inorganic nitrogen. Plant and Soil289, 321e334.

Edwards, A.C., Scalenghe, R., Freppaz, M., 2007. Changes in the seasonal snow coverof alpine regions and its effect on soil processes: a review. Quaternary Inter-national 162, 172e181.

Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Toward an ecological classification ofsoil bacteria. Ecology 88, 1354e1364.

Fierer, N., Jackson, R.B., 2006. The diversity and biogeography of soil bacterialcommunities. Proceedings of the National Academy of Sciences of the UnitedStates of America 103, 626e631.

Frankenberger Jr., W.T., Johanson, J.B., 1982. Effect of pH on enzyme stability in soils.Soil Biology & Biochemistry 14, 433e437.

Geisseler, D., Horwath, W., Scow, K., 2011. Soil moisture and plant residue additioninteract in their effect on extracellular enzyme activity. Pedobiologia 54, 71e78.

Gutknecht, J.L.M., Field, C.B., Balser, T.C., 2012. Microbial communities and theirresponses to simulated global change fluctuate greatly over multiple years.Global Change Biology 18, 2256e2269.

Heisler-White, J.L., Knapp, A.K., Kelly, E.F., 2008. Increasing precipitation event sizeincreases aboveground net primary productivity in a semi-arid grassland.Oecologia 158, 129e140.

Henry, H.A., Juarez, J.D., Field, C.B., Vitousek, P.M., 2005. Interactive effects ofelevated CO2, N deposition and climate change on extracellular enzyme activityand soil density fractionation in a California annual grassland. Global ChangeBiology 11, 1808e1815.

Hobbie, E.A., Jumpponen, A., Trappe, J., 2005. Foliar and fungal 15N: 14N ratios reflectdevelopment of mycorrhizae and nitrogen supply during primary succession:testing analytical models. Oecologia 146, 258e268.

Jastrow, J.D., Amonette, J.E., Bailey, V.L., 2007. Mechanisms controlling soil carbonturnover and their potential application for enhancing carbon sequestration.Climatic Change 80, 5e23.

Keeler, B.L., Hobbie, S.E., Kellogg, L.E., 2009. Effects of long-term nitrogen additionon microbial enzyme activity in eight forested and grassland sites: implicationsfor litter and soil organic matter decomposition. Ecosystems 12, 1e15.

Kemmitt, S.J., Wright, D., Goulding, K.W., Jones, D.L., 2006. pH regulation of carbonand nitrogen dynamics in two agricultural soils. Soil Biology & Biochemistry 38,898e911.

Khare, N., Hesterberg, D., Martin, J.D., 2005. XANES investigation of phosphatesorption in single and binary systems of iron and aluminum oxide minerals.Environmental Science and Technology 39, 2152e2160.

Knapp, A.K., Fay, P.A., Blair, J.M., Collins, S.L., Smith, M.D., Carlisle, J.D., Harper, C.W.,Danner, B.T., Lett, M.S., McCarron, J.K., 2002. Rainfall variability, carbon cycling,and plant species diversity in a mesic grassland. Science 298, 2202e2205.

LeBauer, D.S., Treseder, K.K., 2008. Nitrogen limitation of net primary productivityin terrestrial ecosystems is globally distributed. Ecology 89, 371e379.

Liu, W., Zhang, Z., Wan, S., 2009. Predominant role of water in regulating soil andmicrobial respiration and their response to climate change in a semiaridgrassland. Global Change Biology 15, 184e195.

Liu, X., Zhang, Y., Han, W., Tang, A., Shen, J., Cui, Z., Vitousek, P., Erisman, J.W.,Goulding, K., Christie, P., Fangmeier, A., Zhang, F., 2013. Enhanced nitrogendeposition over China. Nature 494, 459e462.

Milcu, A., Thebault, E., Scheu, S., Eisenhauer, N., 2010. Plant diversity enhances thereliability of belowground processes. Soil Biology & Biochemistry 42,2102e2110.

Miller, M., Paloj€arvi, A., Rangger, A., Reeslev, M., Kjøller, A., 1998. The use of fluo-rogenic substrates to measure fungal presence and activity in soil. Applied andEnvironmental Microbiology 64, 613e617.

Moorhead, D.L., Sinsabaugh, R.L., 2006. A theoretical model of litter decay andmicrobial interaction. Ecological Monographs 76, 151e174.

N€asholm, T., Ekblad, A., Nordin, A., Giesler, R., H€ogberg, M., H€ogberg, P., 1998. Borealforest plants take up organic nitrogen. Nature 392, 914e916.

Niu, S., Yang, H., Zhang, Z., Wu, M., Lu, Q., Li, L., Han, X., Wan, S., 2009. Non-additiveeffects of water and nitrogen addition on ecosystem carbon exchange in atemperate steppe. Ecosystems 12, 915e926.

Parham, J.A., Deng, S.P., 2000. Detection, quantification and characterization of b-glucosaminidase activity in soil. Soil Biology & Biochemistry 32, 1183e1190.

Pendall, E., Rustad, L., Schimel, J., 2008. Towards a predictive understanding ofbelowground process responses to climate change: have we moved any closer?Functional Ecology 22, 937e940.

Ramirez, K.S., Craine, J.M., Fierer, N., 2010. Nitrogen fertilization inhibits soil mi-crobial respiration regardless of the form of nitrogen applied. Soil Biology &Biochemistry 42, 2336e2338.

Ramirez, K.S., Craine, J.M., Fierer, N., 2012. Consistent effects of nitrogen amend-ments on soil microbial communities and processes across biomes. GlobalChange Biology 18, 1918e1927.

Rousk, J., Brookes, P.C., Bååth, E., 2009. Contrasting soil pH effects on fungal andbacterial growth suggest functional redundancy in carbon mineralization.Applied and Environmental Microbiology 75, 1589e1596.

Schimel, J.P., Bennett, J., 2004. Nitrogen mineralization: challenges of a changingparadigm. Ecology 85, 591e602.

R. Wang et al. / Soil Biology & Biochemistry 81 (2015) 159e167 167

Schimel, J.P., Weintraub, M.N., 2003. The implications of exoenzyme activity onmicrobial carbon and nitrogen limitation in soil: a theoretical model. SoilBiology & Biochemistry 35, 549e563.

Sinsabaugh, R.L., Gallo, M.E., Lauber, C., Waldrop, M.P., Zak, D.R., 2005. Extracellularenzyme activities and soil organic matter dynamics for northern hardwoodforests receiving simulated nitrogen deposition. Biogeochemistry 75, 201e215.

Sinsabaugh, R.L., Hill, B.H., Shah, J.J.F., 2009. Ecoenzymatic stoichiometry of mi-crobial organic nutrient acquisition in soil and sediment. Nature 462, 795e798.

Sinsabaugh, R.L., Lauber, C.L., Weintraub, M.N., Ahmed, B., Allison, S.D., Crenshaw, C.,Contosta, A.R., Cusack, D., Frey, S., Gallo, M.E., Gartner, R.B., Hobbie, S.E.,Holland, K., Keeler, B.L., Powers, M.S., Takacs-Vesbach, C., Waldrop, M.P.,Wallenstein, M.D., Zak, D.R., Zeglin, L.H., 2008. Stoichiometry of soil enzymeactivity at global scale. Ecology Letters 11, 1252e1264.

Six, J., Bossuyt, H., Degryze, S., Denef, K., 2004. A history of research on the linkbetween (micro) aggregates, soil biota, and soil organic matter dynamics. Soiland Tillage Research 79, 7e31.

Tabatabai, M.A., 1994. Soil enzymes. In: Mickelson, S.H., Bifham, J.M. (Eds.), Methodsof Soil Analysis. Part 2: Microbiological and Biochemical Properties. Soil ScienceSociety of America, Inc, Madison, WI, pp. 775e833.

Thomas, D.C., Zak, D.R., Filley, T.R., 2012. Chronic N deposition does not alter thebiochemical composition of forest floor and soil organic matter. Soil Biology &Biochemistry 54, 7e13.

Treseder, K.K., Allen, M.F., 2002. Direct nitrogen and phosphorus limitation ofarbuscular mycorrhizal fungi: a model and field test. New Phytologist 155,507e515.

Treseder, K.K., 2008. Nitrogen additions and microbial biomass: a meta-analysis ofecosystem studies. Ecology Letters 11, 1111e1120.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuringsoil microbial biomass-C. Soil Biology & Biochemistry 19, 703e707.

Van Gestel, M., Merckx, R., Vlassak, K., 1996. Spatial distribution of microbialbiomass in microaggregates of a silty-loam soil and the relation with theresistance of microorganisms to soil drying. Soil Biology & Biochemistry 28,503e510.

Vitousek, P.M., Porder, S., Houlton, B.Z., Chadwick, O.A., 2010. Terrestrial phosphoruslimitation: mechanisms, implications, and nitrogen-phosphorus interactions.Ecological Applications 20, 5e15.

Waldrop, M.P., Balser, T.C., Firestone, M.K., 2000. Linking microbial communitycomposition to function in a tropical soil. Soil Biology & Biochemistry 32,1837e1846.

Walker, T.W., Syers, J.K., 1976. The fate of phosphorus during pedogenesis. Geo-derma 15, 1e19.

Wang, R., Filley, T.R., Xu, Z., Wang, X., Li, M.H., Zhang, Y., Luo, W., Jiang, Y., 2014.Coupled response of soil carbon and nitrogen pools and enzyme activities tonitrogen and water addition in a semi-arid grassland of Inner Mongolia. Plantand Soil 381, 323e336.

Wang, A.S., Angle, J.S., Chaney, R.L., Delorme, T.A., McIntosh, M., 2006. Changes insoil biological activities under reduced soil pH during Thlaspi caerulescensphytoextraction. Soil Biology & Biochemistry 38, 1451e1461.

Waring, B.G., Averill, C., Hawkes, C.V., 2013. Differences in fungal and bacterialphysiology alter soil carbon and nitrogen cycling: insights from meta-analysisand theoretical models. Ecology Letters 16, 887e894.

Waring, B.G., Weintraub, S.R., Sinsabaugh, R.L., 2014. Ecoenzymatic stoichiometry ofmicrobial nutrient acquisition in tropical soils. Biogeochemistry 117, 101e113.

Wei, C., Yu, Q., Bai, E., Lü, X., Li, Q., Xia, J., Kardol, P., Liang, W., Wang, Z., Han, X., 2013.Nitrogen deposition weakens plantemicrobe interactions in grassland ecosys-tems. Global Change Biology 19, 3688e3697.

Xu, Z., Wan, S., Zhu, G., Ren, H., Han, X., 2010. The influence of historical land useand water availability on grassland restoration. Restoration Ecology 18,217e225.

Xu, Z., Wan, S., Ren, H., Han, X., Li, M.H., Cheng, W., Jiang, Y., 2012. Effects of waterand nitrogen addition on species turnover in temperate grasslands in northernchina. PLoS ONE 7, e39762.

Zak, D.R., Holmes, W.E., White, D.C., Peacock, A.D., Tilman, D., 2003. Plant diversity,soil microbial communities, and ecosystem function: are there any links?Ecology 84, 2042e2050.

Zeglin, L.H., Stursova, M., Sinsabaugh, R.L., Collins, S.L., 2007. Microbial responses tonitrogen addition in three contrasting grassland ecosystems. Oecologia 154,349e359.

Zhang, N., Liu, W., Yang, H., Yu, X., Gutknecht, J.L.M., Zhang, Z., Wan, S., Ma, K.,2013a. Soil microbial responses to warming and increased precipitation andtheir implications for ecosystem C cycling. Oecologia 173, 1125e1142.

Zhang, S., Li, Q., Lü, Y., Zhang, X., Liang, W., 2013b. Contributions of soil biota to Csequestration varied with aggregate fractions under different tillage systems.Soil Biology & Biochemistry 62, 147e156.

Zhou, X., Chen, C., Wang, Y., Xu, Z., Han, H., Li, L., Wan, S., 2013. Warming andincreased precipitation have differential effects on soil extracellular enzymeactivities in a temperate grassland. Science of the Total Environment 444,552e558.